Department of Urology, The Third Affiliated Hospital of Soochow University, 185 Juqian Street, Changzhou, 213003, China.
BMC Cancer. 2012 Nov 23;12:546. doi: 10.1186/1471-2407-12-546.
A growing body of evidence suggests that microRNAs (miRNAs) play an important role in cancer diagnosis and therapy. MicroRNA-99a (miR-99a), a potential tumor suppressor, is downregulated in several human malignancies. The expression and function of miR-99a, however, have not been investigated in human renal cell carcinoma (RCC) so far. We therefore examined the expression of miR-99a in RCC cell lines and tissues, and assessed the impact of miR-99a on the tumorigenesis of RCC.
MiR-99a levels in 40 pairs of RCC and matched adjacent non-tumor tissues were assessed by real-time quantitative Reverse Transcription PCR (qRT-PCR). The RCC cell lines 786-O and OS-RC-2 were transfected with miR-99a mimics to restore the expression of miR-99a. The effects of miR-99a were then assessed by cell proliferation, cell cycle, transwell, and colony formation assay. A murine xenograft model of RCC was used to confirm the effect of miR-99a on tumorigenicity in vivo. Potential target genes were identified by western blotting and luciferase reporter assay.
We found that miR-99a was remarkably downregulated in RCC and low expression level of miR-99a was correlated with poor survival of RCC patients. Restoration of miR-99a dramatically suppressed RCC cells growth, clonability, migration and invasion as well as induced G1-phase cell cycle arrest in vitro. Moreover, intratumoral delivery of miR-99a could inhibit tumor growth in murine xenograft models of human RCC. In addition, we also fond that mammalian target of rapamycin (mTOR) was a direct target of miR-99a in RCC cells. Furthermore, siRNA-mediated knockdown of mTOR partially phenocopied the effect of miR-99a overexpression, suggesting that the tumor suppressive role of miR-99a may be mediated primarily through mTOR regulation.
Collectively, these results demonstrate for the first time, to our knowledge, that deregulation of miR-99a is involved in the etiology of RCC partially via direct targeting mTOR pathway, which suggests that miR-99a may offer an attractive new target for diagnostic and therapeutic intervention in RCC.
越来越多的证据表明 microRNAs（miRNAs）在癌症的诊断和治疗中发挥着重要作用。miR-99a 是一种潜在的肿瘤抑制因子，在几种人类恶性肿瘤中下调。然而，miR-99a 的表达和功能尚未在人类肾细胞癌（RCC）中进行研究。因此，我们检测了 miR-99a 在 RCC 细胞系和组织中的表达，并评估了 miR-99a 对 RCC 肿瘤发生的影响。
通过实时定量反转录 PCR（qRT-PCR）检测 40 对 RCC 及匹配的相邻非肿瘤组织中 miR-99a 的水平。用 miR-99a 模拟物转染 RCC 细胞系 786-O 和 OS-RC-2，以恢复 miR-99a 的表达。通过细胞增殖、细胞周期、Transwell 和集落形成实验评估 miR-99a 的作用。采用 RCC 小鼠异种移植模型在体内确认 miR-99a 对肿瘤发生的影响。通过 Western blot 和荧光素酶报告基因检测鉴定潜在的靶基因。
我们发现 miR-99a 在 RCC 中显著下调，miR-99a 的低表达水平与 RCC 患者的不良预后相关。恢复 miR-99a 可显著抑制 RCC 细胞的生长、克隆形成、迁移和侵袭，并诱导体外 G1 期细胞周期停滞。此外，miR-99a 的肿瘤内递送可抑制人 RCC 小鼠异种移植模型中的肿瘤生长。此外，我们还发现哺乳动物雷帕霉素靶蛋白（mTOR）是 RCC 细胞中 miR-99a 的直接靶标。此外，siRNA 介导的 mTOR 敲低部分模拟了 miR-99a 过表达的作用，表明 miR-99a 的肿瘤抑制作用可能主要通过 mTOR 调节。
综上所述，这些结果首次表明，miR-99a 的失调部分通过直接靶向 mTOR 通路参与 RCC 的发病机制，这表明 miR-99a 可能为 RCC 的诊断和治疗干预提供有吸引力的新靶点。
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